Protein microscope

ABSTRACT

A system and method for analyzing and imaging a sample containing molecules of interest combines modified MALDI mass spectrometer and SNOM devices and techniques and includes: (A) an atmospheric pressure or near-atmospheric pressure ionization region; (B) a sample holder for holding the sample; (C) a laser for illuminating said sample; (D) a mass spectrometer having at least one evacuated chamber; (E) an atmospheric pressure interface for connecting said ionization region and said mass spectrometer; (F) a scanning near-field optical microscopy instrument; (G) a recording device for recording topography and mass spectrum measurements made during scanning of the sample with the near-field probe; (H) a plotting device for plotting said topography and mass spectrum measurements as separate x-y mappings; and (I) an imaging device for providing images of the x-y mappings.

This application is a continuation of U.S. patent application Ser. No.11/795,687, filed Jul. 20, 2007, which claims the benefit of PCTApplication PCT/US2006/002483, filed Jan. 26, 2006, which claims thebenefit of U.S. Provisional Application No. 60/646,994, filed on Jan.27, 2005, the contents of which are hereby incorporated by reference inits entirety.

BACKGROUND INFORMATION

1. Field of the Invention

The present invention is directed to an instrument for identifyingpeptides, proteins and other biomolecules within a tissue or cell andproviding information on their spatial and temporal distribution. Moreparticularly, this invention relates to an instrument for identifyingpeptides, proteins and other biomolecules within a tissue or cell, whichprovides information on their spatial and temporal distribution down tosubmicron resolution, and which analyzes their activity in vivo.

2. Background of the Invention

Living organisms function through the interactions of biomoleculesintricately distributed in space and varying in time. Often, spatialvariations within a tissue or cell hold the key to understanding thefunction of molecular components. The abundance of biomolecules can spana wide range. For example, protein concentrations range from millimolar(10⁻³ M) to attomolar (10⁻¹⁸ M), and perhaps less. Copy numbers as lowas 10¹ to 10² protein molecules per cell have been reported. Althoughseveral analytical methods offer high sensitivity and spatial resolution(fluorescence measurement, voltammetric microelectrodes, etc.), theselectivity and specificity of these methods seldom allows for theunambiguous identification of biochemical species.

With the emergence of sophisticated mass spectrometric methods, such aselectrospray ionization (ESI) and matrix-assisted laser desorptionionization (MALDI) mass spectrometry, identification and structuralcharacterization of protein and other components has now becomepossible. These techniques offer excellent sensitivity (in certain casesdown to attomolar) as well as detailed biochemical species information(e.g., protein identification including the detection ofpost-translational modifications).

In the mass-spectral analysis of biological materials, however, mostspatial distribution information is lost during the sample preparationstep, wherein cells are ground and thoroughly mixed to form ahomogenized slurry which is then placed in a mass spectrometer foranalysis. The conventional ESI source is not amenable to molecularimaging, as it requires a liquid sample. The situation can be improvedby using MALDI, which involves applying a chemical matrix that islocally excited by laser light so that a plume of sample material isejected from a focused spot on the sample. In principle, MALDI can beused to recover spatial distributions by collecting molecularinformation as the laser is rastered across the sample surface. However,three obstacles exist with the MALDI technique. First, the mixing andco-crystallization of the sample with the light-absorbing matrix largelyobscures the original spatial distribution of analytes (e.g., throughlateral mixing). Second, the need to transfer the sample into a vacuumenvironment for mass analysis considerably restricts the choice ofsamples. Significantly, both of these requirements for successful MALDIanalysis exclude the possibility of in vivo measurements. The thirdobstacle associated with the MALDI technique is that the laws of physicsand practical considerations limit the focusable size of the laser spotso that it is larger than the wavelength of the laser light, resultingin a laser spot larger than most cells of interest and, thereby,diminishing the value of MALDI in view of the need for sampling fromsmaller regions.

Thus, there remains a need in the art for an instrument which can notonly identify peptides and proteins in a tissue or cell but which alsoprovides information on their spatial and temporal distribution down tosubmicron resolution and which analyzes their activity in vivo.

SUMMARY OF THE INVENTION

The present invention is based on the discovery that the combinedcapabilities of scanning near-field optical microscopy (SNOM) imaging,atmospheric pressure-infrared radiation MALDI (AP-IR-MALDI) and massspectrometry will result in a new solution to investigating cellularmaterial down to a ˜100-nm diameter spot. The invention involves theapplication of SNOM techniques in conjunction with mass spectrometry andAP-IR-MALDI to analyze the chemical composition of biological sampleswith significantly greater resolution than previously reported. Thus,the present invention provides a system comprising a mass spectrometer(preferably a time-of-flight mass spectrometer), an AP-IR-MALDIapparatus and a scanning near-field optical microscope (SNOM) which arecombined so as to transform MALDI mass spectrometry into an in vivo“protein microscope” which provides images of biomolecular distributionsin living cells and tissues.

The invention calls for the utilization of a SNOM probe to raster thetip over a sample. Ablated material from individual points is thencollected and analyzed in a highly sensitive mass spectrometer. In thisway, high-spatial-resolution (down to 100 nm scale) maps ofpeptide/protein concentration down to the subcellular level and in realtime are achieved. The improved resolution is achieved by performing thedesorption in the near field zone where the light remains highlycollimated to approximately the width of the aperture.

One aspect of the invention is directed to a system for analyzing andimaging a sample containing molecules of interest, the system having:

(A) an atmospheric-pressure or near-atmospheric-pressure ionizationregion;

(B) a sample holder for holding the sample, the sample holder beingdisposed within said ionization region, the sample comprising an analyteembedded in an ionization-assisting matrix chosen such that said matrixfacilitates ionization of said analyte to form analyte ions uponlight-induced release of said analyte from said sample;

(C) a laser for illuminating said sample, to induce said release of saidanalyte from said sample, and to induce ionization of said analyte toform said analyte ions;

(D) a mass spectrometer having at least one evacuated vacuum chamber andan optional chamber for collision induced dissociation to achievestructural characterization;

(E) an atmospheric pressure interface connecting said ionization regionand said mass spectrometer for capturing said analyte ions released fromsaid sample and for transporting said analyte ions to said spectrometerwith the possibility of additional ionization;

(F) a scanning near-field optical microscopy instrument comprising (a) anear-field probe configured for apertured or apertureless operation forscanning the sample and for focusing the laser light; (b) a vacuumcapillary nozzle, having a heating option, for sucking in particleswhich are desorbed by said laser, the nozzle being connected to an inletorifice of said atmospheric pressure interface; (c) a scanner platformconnected to the sample holder, the platform being movable to a distancewithin a near-field distance of the probe; and (d) a controller formaintaining distance and recording information about a distance betweensaid probe and said sample, to thereby hold said sample within saiddistance; wherein an output of said probe has a spot size on said samplesubstantially equal to an output diameter of said probe;

(G) a means for recording topography and mass spectrum measurements madeduring scanning of the sample with the near-field probe;

(H) a plotting device for plotting said topography and integrated massspectrum measurements as separate x-y mappings; and

(I) means for providing images of the x-y mappings.

In preferred embodiments, the SNOM device has facility for two probes,the second probe being replaced by a vacuum capillary nozzle in ourimplementation. Also in preferred embodiments, the SNOM device isintegrated with an optical microscope, the conventional objective beingreplaced by one suitable for 3 μm wavelength light in ourimplementation.

A second aspect of the invention is directed to a method for performinganalysis and imaging of a sample comprising an analyte embedded in anionization-assisting matrix chosen such that said matrix facilitatesionization of said analyte to form analyte ions upon light-inducedrelease of said analyte from said sample, the method comprising:

-   -   (1) providing the system of this invention;    -   (2) causing the laser to emit light of the proper wavelength and        intensity;    -   (3) projecting the light through the near-field probe to form a        near field zone;    -   (4) positioning the sample to be analyzed within the near-field        zone;    -   (5) irradiating the sample with the light to desorb ions from        the sample;    -   (6) sucking the desorbed ions through the vacuum nozzle and into        the atmospheric pressure interface;    -   (7) causing the desorbed ions and optionally post-ionized        neutrals to enter the mass spectrometer from the atmospheric        pressure interface, to analyze the desorbed ions to determine        the chemical composition of the sample; This step includes an        option to perform collision induced dissociation for structural        characterization;    -   (8) causing the sample to be scanned by the near-field probe;    -   (9) at each pixel, measuring topographical vertical height of        the sample and measuring a complete mass spectrum;    -   (10) causing the measurements made in step (9) to be recorded;    -   (11) causing the recorded topography and mass spectrum        measurements to be plotted as separate x-y mappings; and    -   (12) generating images of the x-y mappings, wherein each color        of each pixel represents an ion intensity value.

In the present invention, the combination of MALDI and SNOM transformsMALDI mass spectrometry into an in vivo “protein microscope”, whichprovides never-before-seen details of the distribution of proteins intissues and living cells, just as the optical microscope has opened upnew worlds by allowing scientists to visualize hitherto invisiblestructures.

The present invention can mitigate and even eliminate the obstaclesassociated with the conventional. MALDI technique.

For example, to replace the artificial organic matrix used in theconventional MALDI technique, the present invention uses mid-infraredlaser radiation for the MALDI imaging. At the proposed wavelength ofapproximately 3 μm, water (the natural medium of living cells) can actas a matrix in the present invention. Thus, tissue sections can beanalyzed directly at the cellular level.

A further aspect of the invention involves analyzing the sample usingatmospheric pressure (AP) MALDI (as opposed to vacuum MALDI), whereinthe sample is mounted in front of the inlet orifice of an AP ion source.The combination of infrared (IR) MALDI with AP MALDI into an AP-IR-MALDIsystem makes in vivo molecular imaging of proteins in cells and tissuesfeasible but with relatively poor spatial resolution.

The third obstacle of the conventional MALDI technique which is overcomeby the present invention is the coarse spatial resolution ofapproximately 10 μm for the proposed IR radiation. This is unacceptablylarge for the study of subcellular structures. To eliminate thisobstacle, the present invention incorporates a scanning near-field(SNOM) imaging system to deliver laser energy with sub-wavelengthresolution. SNOM circumvents the focusing problem of the conventionalMALDI technique by illuminating the sample directly using a sharpenedoptical fiber with an aperture or a metal tip (apertureless SNOM) ratherthan with conventional lenses.

By combining SNOM with AP-IR-MALDI, the present invention enables theunique capability for highly specific in vivo molecular imaging withsub-micron lateral resolution.

In total, the combined capabilities of SNOM, AP-IR-MALDI and massspectrometry result in a new solution to the investigation of cellularmaterial from a spot down to 100-nm in diameter.

The present invention will facilitate high-resolution imaging withframe-by-frame maps of proteins at the sub-cellular level as theorganism responds to different stimuli. The invention will make itpossible, for example, to study the differences between the proteindistributions of healthy cells versus protein distributions of diseasedor medicated cells, the protein content of individual organelles, aswell as a number of other questions having significant scientific,diagnostic and therapeutic implications.

Another advantage of the present invention is that it provides spatiallyresolved chemical analysis of the sample correlated with the surfacetopography. Topographical analysis is achieved by scanning a sharp SNOMprobe across the sample at constant distance from the surface. Chemicalanalysis by means of the laser-induced ionization mass spectrometry isachieved by delivering pulsed laser radiation to the sample surfacethrough the same sharp SNOM probe, and consequent collection andanalysis of mass spectra from the plume generated on the sample by thelaser radiation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of the system of the present invention.

FIG. 2 is a schematic front view of the SNOM instrument, sample holderand scanner platform used in the invention.

FIG. 3 is a schematic diagram of the portion of the system of theinvention composed of the laser, optical fiber, normal force sensor forfeedback, SNOM near-field probe, sample holder/scanner platform andcollection optics and detector. Two versions of the feedback mechanismare shown, a) the optical lever feedback and b) the quartz tuning forkfeedback.

FIG. 4 is a schematic close-up view of the portion of the system of theinvention composed of the ionization region, the SNOM near-field probe,the vacuum capillary nozzle, the sample holder, during the scanning stepof the method of the invention.

FIG. 5 is an atmospheric pressure infrared MALDI mass spectrum of theneuropeptide substance P from liquid water as the matrix. Themid-infrared laser radiation at the wavelength of 2.94 μm was focused onthe target with conventional optics in the far field limit.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a novel system and method for analyzingand imaging samples of interest. The sample, also referred to as thetarget material, normally comprises a mixture of analyte materials andlight-absorbing matrix substances. The sample can be solid or liquid andis composed of one or more materials selected from the groups consistingof peptides, proteins, lipids, carbohydrates, organic compounds andinorganic compounds. A particularly preferred material comprises tissuesamples such as, for example, those disclosed in U.S. Pat. No.5,808,300, which is hereby incorporated by reference herein in itsentirety.

The sample is deposited in a matrix on a target surface of a samplesupport. When illuminated with the laser beam, the matrix molecules areionized and evaporated. The ionized matrix molecules subsequently ionizethe analyte molecules through charge transfer process. At the same time,the analyte molecules, analyte ions and fragmented analyte ions areevaporated together with the matrix ions and molecules.

When the sample to be analyzed and imaged comprises a biologicalmaterial (including living matter), the system and method of thisinvention use an infrared laser and the water content of said materialacting as a matrix.

The system and method of the present invention will be described withreference to FIGS. 1-5 herein.

In FIG. 1, the system of the invention is designated by referencenumeral 1. System 1 includes a sample holder 2 for holding a sample X; aSNOM instrument having a scanning probe 4, which can be implemented inan apertured or an apetureless mode (the latter with the introduction ofan external laser beam 3 via a microscope objective 20 suitable for thelight's wavelength) an independently scannable vacuum capillary nozzle 5and a scanner platform 6 (see FIG. 2); (which is preferablypiezoelectric-actuated); an atmospheric-pressure ornear-atmospheric-pressure ionization region 7; a laser 8; an opticalfiber 9 for optically coupling the laser to the near-field probe; a massspectrometer 10 with an evacuated vacuum chamber 23 and with an optionalchamber for collision induced dissociation 22; an atmospheric pressureinterface 11 connected to the nozzle 5; a controller 24 for controllingthe operation of the SNOM instrument, the mass spectrometer and thelaser, a recording device 25; a plotting device 26; an imaging device27; a data acquisition system 12 and a personal computer 13. System 1further includes a visualization device, e.g., a CCD camera 14, to helppositioning. The camera 14 is connected to a video monitor 15. Thesystem preferably includes an attenuator 16 between laser 8 and fiber 9.

FIG. 2 further shows a reflection detector 17 and a transmissiondetector 18 which can be used in the system and method of the invention.FIG. 3 further shows two version of the normal force sensor system 19for feedback. Version a 19 a is based on feedback from an optical lever,whereas version 19 b uses feedback from a quartz tuning fork.

A sample X of the material to be analyzed is placed on sample holder 2.The sample has a surface facing the tip(s) of the SNOM probe(s). Theposition of the sample relative to SNOM probes 3 and 4 is controlled byscanner platform 6. Scanner platform 6 is preferably a piezoelectriccrystal such as, e.g., that disclosed in U.S. Pat. No. 6,080,586, whichis incorporated by reference herein. Scanner platform may be operated inthe manner disclosed in the aforementioned patent (U.S. Pat. No.6,080,586).

SNOM devices are known in the art. Reference is made, for example, toU.S. Pat. Nos. 6,466,309; 6,080,586; 5,994,691; all of which are herebyincorporated by reference herein.

The SNOM instrument used in the present invention is preferably of atype that allows the simultaneous acquisition of an atomic forcemicroscopy (AFM) signal and a SNOM signal. A piezoelectric-actuatedscanner platform 6 is preferably used for the scanning of the probesrelative to the sample and is used to move the surface of the sampleinto the near-field zone. The scanner platform has either anoptical-lever or a quartz-tuning-fork feedback mechanism for SNOM-tipplacement and has a facility for relative, simultaneous movement of thesample and of the AP-MALDI vacuum nozzle 5 relative to the SNOM probe 3(apertureless) or 4 (apertured). An example of a SNOM instrument meetingthese requirements is commercially available under the designationMultiview 3000™ and manufactured by Nanonics LLP. The MultiView 3000™instrument is described, e.g., at the website http://www.nanonics.co.il,which is hereby incorporated by reference herein.

The particular SNOM instrument for use in the present system is amodified form of conventional SNOM instruments and differs fromconventional SNOM instruments in several ways. First, if an IR laser isused, the SNOM instrument must couple 3-micron infrared light to thesample rather than visible light. To this end, the instrument mustutilize specialty (e.g., chalcogenide, low OH, or sapphire) fibersrather than standard optical fibers. Second, the SNOM instrument usedherein is capable of operation in two modes commonly designated as“apertureless” (wherein laser beam 3 is focused to illuminate a sharpmetal tip 4) and “apertured” (wherein probe 4 is an optical fiber andhas a sub-wavelength integrally-formed aperture at a tip thereof). Inthe apertureless mode, a large (e.g., 10 micron) spot of infrared light3 illuminates a suitably shaped and sharpened metal tip 4 placed a fewnanometers from the sample's surface; the tip enhances the intensity ofthe infrared illumination by a factors of 10 to several thousand in thenear-field zone, depending upon the tip's shape. In the apertured mode,the entire IR excitation is delivered through a sharpened, metallizedfiber which in this case makes up the probe 4. Thus, in the aperturedmode, only the highly-localized, near-field zone is illuminated, but ata slightly lower intensity than in the apertureless mode. One skilled inthe art will select between apertured vs. apertureless mode based uponthe IR-absorptivity of the sample. Third, the SNOM instrument used inthe present invention, unlike conventional SNOM instruments, is fittedwith an independently scannable vacuum nozzle 5 that serves as a part ofthe atmospheric pressure interface.

The SNOM device used in the present invention can have a single probe 4or dual probes. An example of a SNOM device which can be used in thepresent invention is disclosed, e.g., in U.S. Pat. No. 6,080,586, whichis hereby incorporated by reference herein. A preferred dual-probe SNOMdevice for use in the invention is the “MultiView 3000™” instrumentdiscussed above.

The MultiView 3000™ SNOM instrument has two probes in contact with asingle sample with one probe scanning relative to the other. In the SNOMinstrument used in the present invention, one probe can be used toilluminate a precise point on a sample while a sharpened capillaryserving as the vacuum nozzle can replace the second probe.

The system and method of this invention may use a UV or infrared laser8, with the latter being preferred, particularly when the sample to beanalyzed and imaged comprises biological tissue or cells.

The atmospheric-pressure or near-atmospheric-pressure ionization region7 used in the system of this invention is designed to control the gasnature, pressure, temperature, and humidity to maintain the integrity ofthe sample and/or facilitate ionization. In some cases, additionalequipment is incorporated in the ionization region to control theseparameters, such as a heater to control the temperature. The ionizationregion may include a gas inlet as a pathway for gas to enter theatmospherically-controlled region. The ionization region may be filledwith a bath gas at or near atmospheric pressure. The bath gas, which isnormally selected from the group which comprises inert gas, nitrogengas, and gas mixture such as air, is chosen such that it does not reactwith the sample at ambient conditions or even under laser illumination.

If the ionization region is an enclosed structure, e.g., a chamber, itfurther comprises a window through which the illuminating laser beam mayenter in the apertureless mode. The position of the window is correlatedto the position of the sample to be illuminated inside the ionizationchamber. In a preferred embodiment, the window is positioned at the topor bottom of the chamber directly above or below the SNOM tip.

FIG. 4 shows the details of the ionization region of the system.Normally, the sample holder 2 is positioned inside the ionization region(or chamber) so that the deposited sample is close to the inlet orificeof the atmospheric pressure interface between the ionization region andthe spectrometer, and so that the sample is easily illuminated by thelaser beam. The sample holder is normally selected from the groupcomprising insulating materials and conductive materials. If the sampleholder is conductive, it is normally used as an electrode to provide aconstant or time dependent electric field that moves the ionized analytefrom the target surface to the inlet orifice on the AP interface throughwhich the ionized analyte enters the spectrometer. The electric field isgenerated by a constant or time dependent high voltage power supply 21.For the use of time dependent electric fields for the efficient transferof ions into a mass spectrometer, reference is made to U.S. Pat. No.6,791,080 which is hereby incorporated by reference herein. If thesample holder is insulating, a separate electrode behind the sampleholder may be needed to provide the electric field required for iontransportation.

The constant or time dependent electric potential of the inlet orificeand the other electrodes, such as the sample support, is adjusted toachieve the best signal from the spectrometer. The adjustment techniqueis conventional to a person skilled in the art.

The atmospheric pressure (AP) interface 11 used in the system and methodof this invention connects the ionization region 7 to the massspectrometer 10 and entrains the plume of particles (neutrals, ions andparticulates) generated by the laser pulse. The AP interface used in theinvention is modified to accommodate the MALDI source and parts of theSNOM system. In the AP interface, a protruding metal capillary (i.e.,the nozzle 5) is attached to the inlet orifice to facilitate the probingof the laser-generated plume over the sample. Reference is made to U.S.Pat. No. 6,806,468; which is hereby incorporated by reference herein.Capillaries of 10-35 cm in length with an inner diameter ranging from100 μm to 800 μm and with the option of heating are preferably used tomaximize ion transmission efficiency and minimize the gas load on themass spectrometer at the same time. Nozzle 5 collects the laser plumegenerated in the ionization region and directs the plume to the massspectrometer. The heating of the capillary facilitates ion transportinto the mass spectrometer. The AP interface used in the presentinvention differs from commercially available AP interfaces used forelectrospray and MALDI techniques in that the inlet orifice of the APinterface used in the invention has a very small inlet orifice. This isbecause the laser plume generated by the MALDI/SNOM technique in thepresent invention is very small, down to 100 nm in size.

In the AP interface region, the efficient transport and/or collection oflaser-generated ions requires special transfer (e.g., ion guide) and/orstorage (e.g., ion trap) ion optics. In addition, the sensitivity ofdetection can be enhanced by ionizing the entrained neutrals. Thisadditional ionization process (e.g., chemical ionization) takesadvantage of the neutrals desorbed by the laser pulse.

The mass spectrometer 10 used in the system and method of this inventionis preferably an orthogonal acceleration time-of-flight (TOF) massspectrometer because it efficiently combines an atmospheric pressureinterface with the high transmission and high-duty-cycle capabilities ofthe TOF mass spectrometer. Orthogonal acceleration TOF massspectrometers are known in the art. Reference is made, e.g., to U.S.Pat. Nos. 5,117,107 and 6,855,924, which are hereby incorporated byreference herein.

From the collisional damping interface, ions are injected into thesource region perpendicular to the axis of the reflectron type TOF massspectrometer. The pressure in the ion transport region is in the 50 to100 mTorr range. An optional collision cell is placed in the ion beampath. If this cell is filled with collision gas (e.g., argon) the sampleions undergo collision induced dissociation (CID). The resultingfragment ions can be analyzed by the mass spectrometer to producestructural information. Once the ions fill the ejection region, they areaccelerated along the reflectron axis by an ejection pulse of ˜3 kHzfrequency. Ion packets are detected by a dual multichannel platedetector and the corresponding signal is captured by a fast dataacquisition system 12. An example of an orthogonal acceleration TOF massspectometer meeting these requirements is commercially available underthe designation QTOF Premier and manufactured by Waters Co.

In the method of the present invention, the sample holder with attachedsample is placed on the bed of the sample scanner, and the SNOM head ispositioned over the top of the sample. The IR laser beam is injectedinto the optical fiber lithe SNOM probe is apertured or focused on themetal tip if the probe is apertureless. For SNOM instruments utilizingoptical feedback, the optical cantilever system is aligned to produce amaximum signal from a four-quadrant photodiode (e.g., 2 volts or more).The mechanical oscillator frequency is tuned to a resonance of the SNOMfiber. The preferred frequency is between 20 and 100 kHz and the peakshould have a signal-to-noise value of at least 25.

The SNOM near-field probe 3 is lowered to the sample surface in theautomatic mode. Once the probe tip 3A comes to close contact with thesample, the vertical servo motor will shut off, and automatic-feedbackpositioning commences. The position of nozzle 5 and nozzle tip 5A areadjusted to attain the maximum signal on the mass spectrometer. Thesample is scanned in the horizontal plane beneath the SNOM probe tip13A. At each pixel, the topographical vertical height of the sample anda complete mass spectrum are measured. These measurements are recordedby the computer controller and the topography and mass spectrum areplotted, in real time, as separate x-y mappings in which the color ofeach pixel on the computer monitor screen represents the value of theintensity of the particular ions selected.

With the sample and the SNOM probe in place, an appropriate voltagedifference (e.g., up to 3 kV) is applied between the sample holder andthe collection nozzle. The transfer capillary is heated to ˜100° C. tominimize adsorption of the particles to its wall. As noted previously,the mass spectrometer used in the present invention is preferably anorthogonal acceleration TOF mass spectrometer which has been modified asdescribed hereinabove. The ion transfer optics, accelerating and ionfocusing voltages and the injection times are optimized for the best ionsignal. At the current state of technology, the data acquisition rate islimited by the repetition rate of the laser. As the mid-IR laser sourcesin the required ˜10 mJ/pulse energy range are available with a maximumrepetition rate of 10 Hz, the spectrum collection from a surface point,even under ideal conditions, requires 100 ms.

Once a mass spectrum with at least a signal-to-noise ratio of 3 iscollected at a particular location on the sample, the sample is moved tothe next interrogation point. Such a mass spectrum is shown in FIG. 5.This spectrum was collected using said atmospheric pressure interface11, on a QTOF Premier mass spectrometer using mid-infrared laserradiation at 2.94 μm in the far field limit. The sample, neuropeptidesubstance P, was desorbed and ionized from liquid water as the matrix.The signal-to-noise ratio can be improved by spectrum averagingtechniques. Identification of an unknown species can be facilitated bythe introduction of a collision chamber between the ion transfer opticsand the orthogonal acceleration region. Elevating the pressure of, forexample, argon gas in this collision chamber results in fragmentation ofthe primary ions. Detecting these fragment ions can contribute to theidentification of the primary ions.

Following the scan, the data are analyzed by producing false colormappings of the intensity of selected peaks. Similarly, to the spectraand height data plotted during the measurements, the color of each pixelin the image of the sample is determined by the intensity of the peak ofinterest at that position on the sample.

As mentioned above, the present invention provides spatially resolvedchemical analysis of the sample correlated with the surface topography.Topographical analysis is achieved by scanning a sharp SNOM probe acrossthe sample at constant distance from the surface. Chemical analysis bymeans of the laser-induced ionization mass spectrometry is achieved bydelivering pulsed laser radiation to the sample surface through the samesharp SNOM probe, and consequent collection and analysis of mass spectrafrom the plume generated on the sample by the laser radiation. Thechemical and topographical analysis may be carried out, for example, inthe manner taught in U.S. Pat. No. 6,466,309.

EXPERIMENTAL

This example illustrates an attempt to define the surface proteome (orinternal cellular components including cytoplasmic and/or nuclear),using securely attached cells. Cell tissues or non-adherent cells mayrequire specific ligands to securely attach live cells to slides priorto use in the system of this invention. In this example, samples areprepared using multiple reagents for cell attachment, including use ofcoated, inorganic substances (e.g., glass, silicon, mica). The slidesmay contain any of the established attachment chemicals including,polystyrene, 3-aminopropylsilane, poly-L-lysine, poly(ethylene-imine)(PEI), polyethylene glycol) (PEG) photolithography, cell-adhesiveligands, chitosan, fibronectin, laminin, collagen type I, or specificantibodies (i.e., anti-CD5 or anti-CD19 antibodies), to engineerspecific cell-surface interactions within the individual slide areas.

For most non-living biological samples (e.g., tissue sections), near-UVlight can be used for ion generation. This is significant when definingspecific protein/nucleic acid interactions, since illumination at both365 and 410 nm results in significant cross-linking of proteins with theDNA. As an alternative, a non-invasive live-cell measurement of changesusing long-wavelength light (i.e., 710 nm) may be used. However, formost living biological samples, it is preferred to use focused,near-infrared laser light scanned across a field of the cell. Theresponse of the cell to the laser may depend on its size, structure,morphology, composition, and surface membrane properties. Viability ofcells is determined by means of molecular probes for fluorescencemicroscopy (Live/Dead kit-double staining with calcein AM and ethidiumhomodimer). Both UV and IR methods described here will allowquantitative measurements of cellular components with their functionalparameters intact.

In this example, the eggs of the African clawed frog Xenopus laevis arestudied. These eggs are relatively large in size (˜1 mm), thus providean excellent test case for live imaging. Analyte loading in a singleXenopus egg is calculated based on the average concentration of variousproteins. For example, the proteins p13Suc and Cdc25 are present at 2.5μM and 0.14 μM concentrations in the eggs, respectively. The volume of atypical egg is ˜0.9 μL (the diameter is between 1.0 and 1.3 mm). Thus,putting a single egg on the sample holder results in loadings of p13Sucand Cdc25 of 2 μmol and 0.1 pmol, respectively. The demonstrateddetection limit for small peptides using AP-MALDI with IT-MS is in thelow femtomole range.

Other proteins that may be present on the cell surface of most cells,including those of the Xenopus egg, include:

ATP binding DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 1, sodium-potassium-transporting ATPase Ca+ binding Annexin I, annexin V, annexinVI, annexin A2, calreticulin, alpha-actin 1 Chaperone Cyclophilin A,glucose-regulated protein, heat shock protein 70 kDa, heat shock protein90 kDa, Tumor rejection antigen Cytoskeleton a-Actin, b-actin, cofilin,filamin, keratin, moesin, a-tubulin, b-tubulin, vimentin (actin sizesare 41-42 KD) Enzymes Acminopeptidase N, transketolase, vinculinGlycolysis Anolase, glyceraldehydes-3-phosphate dehydrogenase, lactatedehydrogenase, phosphoglycerate kinase, serine/threonine proteinphosphatase PP1, triosephosphate isomerase Protein transportADP-ribosylation factor 4, clathrin, polyubiquitin 3, b-RAB GDPdissociation inhibitor, Ca and Cl channels Signal transduction a1-Caseinkinase 2, 14-3-3 protein (four isoforms), receptor tyrosine kinasesTranscription-translation Eukaryotic translation elongation factor 2,eukaryotic translation elongation factor 1, enhancer protein, eukaryotictranslation initiation factor 4A

In the present example, the substrate with attached sample is placed onthe bed of the sample scanner, and the SNOM head is positioned over thetop. The IR laser beam is injected into the optical fiber for theapertured mode, or focused on the metal tip for the apertureless mode.The optical cantilever system is aligned to produce a maximum signalfrom the four-quadrant photodiode (2 volts or more), and the mechanicaloscillator frequency is tuned to a resonance of the SNOM fiber (thepreferred frequency is between 20 and 100 kHz, the peak should have anintensity a signal-to-noise value of at least 25). The effective forceat the peak is noted and the control force is set about 30% lower thanthis value). Next the SNOM head is lowered to the sample surface in theautomatic mode. Once the tip comes to close contact, the vertical servomotor is shut off, and automatic-feedback positioning commences. TheMALDI nozzle position is adjusted to attain the maximum signal on themass spectrometer. The sample is scanned in the horizontal plane beneaththe SNOM tip. With the sample and the SNOM head in place, an appropriatevoltage is applied to the sample holder (e.g., 2 kV) and the transfercapillary is heated to ˜100° C. to minimized adsorption of the particlesto its wall. An orthogonal acceleration TOF mass spectrometer is used.The ion transfer optics, accelerating and ion focusing voltages and theinjection times are optimized for best ion signal.

Once a mass spectrum with at least a signal-to-noise ratio of 3 iscollected at a particular location on the sample (see, e.g., FIG. 5),the sample is moved to the next interrogation point.

At each pixel, the topographic vertical height of the sample and acomplete mass spectrum are measured. These are recorded by a computercontroller and the topography and mass spectrum are plotted, in realtime, as separate x-y mappings in which the color of each pixel on thescreen represents the value of the intensity of the particular ionselected.

Following the scan, the data are analyzed by producing false colormappings of the intensity of selected peaks. Similarly to the spectraand height data plotted during the measurements, the color of each pixelin the image of the sample is determined by the intensity of the peak ofinterest at that position on the sample.

The foregoing description of the invention is thus illustrative andexplanatory, and various changes in the equipment, as well as in thedetails of the methods and techniques disclosed herein may be madewithout departing from the spirit of the invention, which is defined bythe claims.

1. A system for analyzing and imaging a sample containing molecules ofinterest, the system having: (A) an atmospheric-pressure ornear-atmospheric-pressure ionization region; (B) a sample holder forholding the sample, the sample holder being disposed within saidionization region, the sample comprising at least one analyte embeddedin an ionization-assisting matrix such that said matrix facilitatesionization of said analyte forms analyte ions upon light-induced releaseof said analyte from said sample; (C) a laser for near fieldillumination of said sample, to induce said release of said analyte fromsaid sample, and to induce ionization of said analyte to form saidanalyte ions; (D) a mass spectrometer having at least one evacuatedvacuum chamber; (E) an atmospheric pressure interface connecting saidionization region and said mass spectrometer (a) for capturing saidanalyte ions and neutrals released from said sample, (b) for subjectingthe neutrals to additional ionization or post ionization, and (c) fortransporting said analyte ions to said spectrometer; and (F) a scanningnear-field optical microscopy instrument comprising (a) a near-fieldapertureless probe in which said laser is focused onto a sharp metal ormetalized tip for scanning the sample; (b) a vacuum capillary nozzle forsucking in particles which are ejected by said laser, the nozzle beingconnected to an inlet orifice of said atmospheric pressure interface;(c) a scanner platform connected to the sample holder, the platformbeing movable to a distance within a near-field distance of the probe;and (d) a controller for maintaining distance information about adistance between said probe and said sample, to thereby hold said samplewithin said distance; wherein an output of said probe has a spot size onsaid sample roughly equal to the output diameter of said probe.
 2. Asystem according to claim 1, wherein the scanning near-field probehaving a sub-wavelength apex integrally formed at a tip thereof, thescanning near-field optical microscopy instrument further comprises afiber optic cable for carrying light from the laser.
 3. A systemaccording to claim 1, wherein the laser is an infrared laser.
 4. Asystem according to claim 1, wherein the scanning near-field opticalmicroscopy instrument comprises facility for two probes for scanning thesample, wherein a first probe is used to illuminate a precise point on asample while a second probe is modified to a vacuum capillary nozzlewith heating for collecting analyte ions, and further comprising apositioning device for independently controlling the position of thefirst probe and the second probe.
 5. A system according to claim 1,wherein the mass spectrometer is time-of-flight mass spectrometer.
 6. Asystem according to claim 1, further comprising a time dependent highvoltage power supply for generating a time dependent electric field fortransporting said analyte ions to said atmospheric pressure interface,said electric field having electric pulses which are applied directly tothe vacuum capillary nozzle.
 7. The system of claim 1, wherein saidlaser operates at a 2.94 micrometer wavelength.
 8. The system of claim1, wherein said mass spectrometer further comprises a chamber forcollision induced dissociation to achieve structural characterization.9. The method of claim 1, further comprising a recording device forrecording topography and mass spectra made during scanning of the samplewith the near-field probe.
 10. The method of claim 1, further comprisinga plotting device for plotting topography and mass spectrum measurementsas separate x-y mappings.
 11. The method of claim 1, further comprisingan imaging device for providing images of x-y mappings of topography andmass spectrum measurements.
 12. A method for performing analysis andimaging of a sample comprising an analyte embedded in anionization-assisting matrix, whereby said matrix facilitates ionizationof said analyte to form analyte ions upon light-induced release of saidanalyte from said sample, the method comprising: (1) providing a systemfor analyzing and imaging a sample, the system having: (A) anatmospheric-pressure or near-atmospheric-pressure ionization region; (B)a sample holder for holding the sample, the sample holder being disposedwithin said ionization region, the sample comprising an analyte embeddedin an ionization-assisting matrix chosen such that said matrixfacilitates ionization of said analyte to form analyte ions uponlight-induced release of said analyte from said sample; (C) a laser forilluminating said sample, to induce said release of said analyte fromsaid sample, and to induce ionization of said analyte to form saidanalyte ions; (D) a mass spectrometer having at least one evacuatedvacuum chamber; (E) an atmospheric pressure interface connecting saidionization region and said mass spectrometer for capturing said analyteions released from said sample, subjecting the neutrals to additionalionization or post ionization, and for transporting said analyte ions tosaid spectrometer; and (F) a scanning near-field optical microscopyinstrument comprising (a) a near-field probe for scanning the sample;(b) a vacuum capillary nozzle for sucking in particles which are ejectedby said laser, the capillary nozzle being connected to an inlet orificeof said atmospheric pressure interface; (c) a scanner platform connectedto the sample holder, the platform being movable to a distance within anear-field distance of the probe; and (d) a controller for maintainingdistance information about a current distance between said probe andsaid sample, to thereby hold said sample within said distance; whereinan output of said probe has a spot size on said sample roughly equal toan output diameter of said probe; (2) causing the laser to emit light ofthe proper wavelength and intensity; (3) projecting the light onto thenear-field probe to form a near field zone; (4) positioning the sampleto be analyzed within the near-field zone; (5) irradiating the samplewith the light to eject ions and neutrals from the sample; (6)subjecting the neutrals to additional ionization or post ionization; (7)sucking the ejected ions and neutrals through the vacuum nozzle and intothe atmospheric pressure interface; (8) causing the produced ions andneutrals to enter the mass spectrometer from the atmospheric pressureinterface; (9) causing the sample to be scanned relative to thenear-field probe; and (10) at each pixel, measuring topographicalvertical height of the sample and measuring a complete mass spectrum.13. A method according to claim 12, wherein the sample consists of ananalyte embedded in the ionization-assisting matrix, and the analyte insaid sample comprises at least one material selected from the groupsconsisting of peptides, proteins, lipids, carbohydrates, metabolites,organic compounds and inorganic compounds.
 14. A method according toclaim 12, wherein the sample consists of at least one analyte embeddedin the ionization-assisting matrix, and the analyte comprises abiological material comprising living matter, further wherein the matrixcomprises the water content of the sample.
 15. A method according toclaim 14, wherein the laser used in the method is an infrared laser. 16.A method according to claim 12, wherein said additional ionization ischemical ionization.
 17. A method according to claim 12, wherein thematrix comprises the water content of the sample and the laser used inthe method is an infrared laser.
 18. A method according to claim 12,further comprising applying a time dependent electric field to transportsaid produced ions to the atmospheric pressure interface, said electricfield having electric pulses which are applied directly to the vacuumcapillary nozzle.
 19. The method of claim 12, wherein said laseroperates at a 2.94 micrometer wavelength.
 20. The method according toclaim 12, wherein said additional ionization is electrospray ionization.21. The method of claim 12, wherein the mass spectrometer furthercomprises a chamber for collision induced dissociation to achievestructural characterization.
 22. The method of claim 12, wherein themass spectrometer further comprises a post ionization region, and saidmethod comprises postionizing the neutrals at the postionization region.23. The method of claim 12, wherein said step of causing the producedions and neutrals to enter the mass spectrometer, fragment in acollision cell and to analyse them to determine the chemical compositionof the sample.
 24. The method of claim 12, further comprising an imagingdevice for providing images of x-y mappings of topography and massspectrum measurements.
 25. The method of claim 12, further comprisingcausing the measurements made in step (10) to be recorded.
 26. Themethod of claim 12, further comprising generating images of the x-ymappings, wherein each color of each pixel represents an ion intensityvalue.
 27. The method of claim 12, further comprising a recording devicefor recording topography and mass spectrum measurements made duringscanning of the sample with the near-field probe.